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Query: UNIPROT:P01034 (cystatin C)
 3,397 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The contribution of the kininogens and cystatin C to the functional inhibitory capacity for cysteine proteinases of blood plasma and inflammatory secretions was estimated from ex vivo experiments. 98.5% of the inhibitory capacity of blood plasma for cathepsin L (4-5 microM) is provided by the kininogens ensuring a complete control of this enzyme even at a lowered kininogen concentration. Control of cathepsin B activity by the kininogens is incomplete and depends critically on the active concentration of cystatin C (70 nM in normal plasma), which is reduced in blood plasma of polytraumatized and septic patients and very low in epithelial lining fluid of the shock lung.
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PMID:The role of the kininogens as cysteine proteinase inhibitors in local and systemic inflammation. 146 82

An inhibitory dodecameric peptide was designed which tentatively mimics the inhibitory site of cystatin C-like structures. Succinylated and mansylated derivatives were also synthesised and assayed for their inhibiting properties towards papain and rat cathepsins B, H and L. All peptides preferentially inhibit cathepsin L and papain as their naturally occurring inhibitor model. A significant increase in inhibition was obtained after mansylation of the crude peptide with Ki values in the micromolar or 0.1 micromolar range. The use and interest of such peptide inhibitors are discussed.
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PMID:Simulation of the inhibitory cystatin surface by a synthetic peptide. 231 Mar 84

Two cysteine proteinase inhibitors of the cystatin C type have been purified from urine of sodium chromate-treated rats. Both strongly inhibit papain as well as rat liver cathepsin L (Ki less than 10(-11) M) whereas rat liver cathepsins B and H are inhibited to a lesser extent. They differ by their apparent molecular mass of 17 kDa and 22 kDa and by their isoelectric point greater than or equal to 9.5 and 7.7 respectively. These two molecules share complete immunochemical identity and are precipitated by antibodies directed against human cystatin C but not by anti rat thiostatin and anti rat H-kininogen antibodies. They are also found in large amounts in seminal vesicles where they represent most of the cysteine proteinase inhibitory capacity.
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PMID:Purification of the cystatin C-like inhibitors from urine of nephropathic rats. 314 92

Procathepsin L, the precursor to a powerful lysosomal cysteine proteinase, has been purified to apparent homogeneity from guinea pig spermatozoa, a novel and previously unrecognized source of this catalytically active zymogen. In the range of pH 5.0, procathepsin L (39,000 M(r)) readily self-processed yielding a mature, single-chain proteinase (29,000 M(r)) and an intact propeptide (10,000 M(r)) by what appeared kinetically to be an intramolecular reaction mechanism. These characteristics resembled those reported for the "major excreted protein" (MEP) of malignantly transformed mouse fibroblasts-a protein that has been characterized as the precursor to the mouse analog of human cathepsin L (B. R. Troen, S. Gal, and M. M. Gottesman (1987) Biochem. J. 246, 731-735). Other characteristics shared by the guinea pig and mouse zymogens included proteolytic activity at pH 5.0, homologous N-terminal amino acid sequences, and immunological relatedness. It was thus concluded that acrosomal procathepsin L is the guinea pig analog of MEP. Acrosomal procathepsin L had a specific activity on benzyloxy-carbonyl-Phe-Arg-7-(4-methyl)coumarylamide (Z-Phe-Arg-NMec) of 30 mumol min-1 mg-1 enzyme at pH 3.2 and 37 degrees C. Relative to the assay substrate, rates on other fluorogenic substrates were 90% for Z-Phe-Cit-NMec, 63% for Z-Leu-Leu-Arg-NMec, 43% for D-Phe-Ser(Bzl)-Phe-Phe-Ala-Ala-p-aminobenzoate (a "specific" cathepsin D assay substrate), and 32% for Z-Val-Val-Arg-NMec. No action was detected on Z-Arg-Arg-NMec or Arg-NMec. Mature cathepsin L showed the same relative order of substrate specificity as its proenzyme form, but the absolute rates were about 5-fold greater. Additionally, the mature (single-chain) form of cathepsin L displayed Km and kcat values on Z-Phe-Arg-NMec that yielded an exceptionally high catalytic coefficient (11,600 s-1 mM-1) compared to values reported for two-chain forms of cathepsin L. Self-processing by acrosomal procathepsin L at pH 5.5 was totally inhibited by leupeptin, cystatin C, Ep-475, and Z-Phe-Phe-CHN2 at 1 microM levels. Gossypol (0.1 mM) gave 94% inhibition. Interestingly, dextran sulfate (100 micrograms ml-1) gave a 3.6-fold increase in the rate of self-processing seen at pH 5.5--a phenomenon of potential physiological relevance in view of the high-negative-charge density present within the hyaluronic acid-rich outer layer (cumulus oophorus) of the ovum.
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PMID:Purification and characterization of procathepsin L, a self-processing zymogen of guinea pig spermatozoa that acts on a cathepsin D assay substrate. 748 6

The near-UV spectroscopic changes induced by the binding of recombinant human cystatin A to papain were appreciably different from those induced by cystatin C, reflecting mainly interactions involving the single tryptophan of cystatin C, Trp-106. Cystatin A bound tightly and rapidly to papain and cathepsin L, with dissociation equilibrium constants of approximately 10(-11)-10(-13) M and association rate constants of 3 x 10(6)-5 x 10(6) M-1.s-1. These affinities are at least 50-100-fold higher than previously reported values. The kinetics of binding to papain were consistent with a simple reversible bimolecular reaction mechanism, indicating that cystatin A, like chicken cystatin and cystatin C, binds to papain with no appreciable conformational adaptation of either reacting protein. Cystatin A bound more weakly to actinidin and cathepsins B, C and H, with dissociation equilibrium constants of 10(-8)-10(-9) M. The weaker binding to cathepsin B was largely due to a considerably reduced association rate constant (approximately 4 x 10(4) M-1.s-1), consistent with the 'occluding loop' of cathepsin B markedly restricting the access of cystatin A to the active site. The lower affinities for actinidin and cathepsins C and H were due partly to lower association rate constants (2 x 10(5)-6 x 10(5) M-1.s-1) but primarily to higher dissociation rate constants. The mode of binding of cystatin A to inactivated papains indicated that there is appreciably less space around the active-site cysteine of papain in the complex with cystatin A than in the complexes with chicken cystatin and cystatin C. An N-terminally truncated form of cystatin A, lacking the first six residues, had considerably lower affinity for papain than the full-length inhibitor, consistent with an intact N-terminal region being of importance for proteinase binding.
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PMID:Characterization by spectroscopic, kinetic and equilibrium methods of the interaction between recombinant human cystatin A (stefin A) and cysteine proteinases. 757 65

The kinetics of pH-induced inactivation of human cathepsins B and L was studied by conventional and stopped-flow methods. The inactivation of both enzymes was found to be an irreversible, first-order process. The inactivation rate constants increased exponentially with pH for both enzymes. From log kinac vs pH plots, 3.0 and 1.7 protons were calculated to be desorbed for pH-induced inactivation of cathepsins L and B. Cathepsin B was thus substantially more stable than cathepsin L (approximately 15-fold at pH 7.0 and 37 degrees C). Cathepsin B was efficiently inhibited by cystatin C at pH 7.4, whereas the inhibition by stefin B and high molecular weight kininogen was only moderate. In contrast, cathepsin L was efficiently inhibited by both chicken cystatin and stefin B at this pH kass approximately 3.3 x 10(7) m-1 s-1).
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PMID:Regulation of the activity of lysosomal cysteine proteinases by pH-induced inactivation and/or endogenous protein inhibitors, cystatins. 762 31

The structural basis for the biological specificity of human cystatin C has been investigated. Cystatin C and other inhibitors belonging to family 2 of the cystatin superfamily interact reversibly with target peptidases, seemingly by independent affinity contributions from a wedge-shaped binding region built from two loop-forming inhibitor segments and a binding region corresponding to the N-terminal segment of the inhibitor. Human cystatin C variants with Gly substitutions for residues Arg-8, Leu-9, and/or Val-10 of the N-terminal binding region, and/or the evolutionarily conserved Trp-106 in the wedge-shaped binding region, were produced by site-directed mutagenesis and Escherichia coli expression. A total of 10 variants were isolated, structurally verified, and compared to wild-type cystatin C with respect to inhibition of the mammalian cysteine peptidases, cathepsins B, H, L, and S. Varying contributions from the N-terminal binding region and the wedge-shaped binding region to cystatin C affinity for the four target peptidases were observed. Interactions from the side chains of residues in the N-terminal binding region and Trp-106 are jointly responsible for the major part of cystatin C affinity for cathepsin L and are also of considerable importance for cathepsin B and H affinity. In contrast, for cathepsin S inhibition these interactions are of lesser significance, as reflected by a Ki value of 10(-8) M for the cystatin C variant devoid of Arg-8, Leu-9, Val-10, and Trp-106 side chains. The side chain of Val-10 is responsible for most of the affinity contribution from the N-terminal binding region, for all four enzymes. The contribution of the Arg-8 side chain is minor, but significant for cystatin C interaction with cathepsin B. The Leu-9 side chain confers selectivity to the inhibition of the target peptidases; it contributes to cathepsin B and L affinity by factors of 200 and 50, respectively, to cathepsin S binding by a factor of 5 only, and results in a 10-fold decreased affinity between cystatin C and cathepsin H.
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PMID:Structural basis for the biological specificity of cystatin C. Identification of leucine 9 in the N-terminal binding region as a selectivity-conferring residue in the inhibition of mammalian cysteine peptidases. 789 Jun 20

We investigated the appearance and activity of the cysteine proteinase cathepsin B and its physiological inhibitors, stefins A and B, at the cellular level in human tumor cell lines HS-24, derived from a primary lung tumor (squamous cell), and SB-3, derived from a metastasis (lung adenocarcinoma). In addition to cathepsin B, these tumor cells also expressed the immunologically and functionally related cathepsin L, but not cathepsin H. Stefin A was found in HS-24 but not in SB-3 cells; stefin B was found in both cell types. Using a specific fluorogenic cytochemical assay, the intracellular activity of the enzyme was localized and quantified. Thus, the cellular cathepsin B kinetics for the synthetic substrates Z-Arg-Arg-4M beta NA and Z-Val-Lys-Lys-Arg-4M beta NA, its pH dependence and inhibition by E64, stefins A and B, and cystatin C could be determined. From these measurements it appeared that the enzyme exhibited different cleavage rates for these substrates in the different cell types, showed considerable cleavage activity at neutral pH, which was stable under these conditions for extended time periods, and was highly sensitive to the inhibitors E64 and cystatin C but was considerably less sensitive to stefins, particularly stefin A. By conventional light microscopy, confocal laser scanning microscopy, and electron microscopy the enzymatic activity was localized in lysosomes, as expected, but also in the endoplasmic reticulum, nuclear membrane, and plasma membrane. The endoplasmic reticulum is a site at which only pre-mature enzyme forms exist, which are usually not active. The appearance of enzymatic activity at the plasma membrane confirms earlier biochemical and immunofluorescence microscopic investigations. The different sites of localization within the cells make it likely that different forms of the enzyme are expressed simultaneously, which follow alternate ways of processing and sorting. Taken together, the results support an involvement of the enzyme under extracellular conditions in degradative processes.
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PMID:Cathepsin B activity in human lung tumor cell lines: ultrastructural localization, pH sensitivity, and inhibitor status at the cellular level. 801 75

Human cystatin D is a novel member of the cystatin superfamily of cysteine proteinase inhibitors present in saliva and tears. Two alleles of the cystatin D gene (CST5), encoding protein variants with either Cys or Arg as residue 26 in their 122-residue polypeptide chains, are present in the population. Expression of the two alleles was investigated by immunochemical analyses of the secreted cystatin D in saliva from individuals homozygous for each of the two alleles, with results demonstrating that both are expressed at similar levels. The inhibitory characteristics of the two cystatin D variants were studied, by determination of dissociation equilibrium constants (Ki) for their complexes with papain and with the mammalian cysteine proteinases, cathepsins B, H, L, and S. The results demonstrate that 1) cystatin D has a characteristic inhibition profile since it does not inhibit cathepsin B (Ki > 1 microM), and when compared to cystatin C and all other known cystatins it is a much poorer inhibitor of cathepsin L (mean Ki 25 nM) but binds cathepsin H and S relatively tightly (mean Ki values of 8.5 and 0.24 nM, respectively); and 2) the inhibitory activities of the two cystatin D variants are not significantly different, demonstrating that the presence of an extra cysteine residue in the cystatin D molecule affects neither the stability nor the functional activity of the inhibitor, thus explaining the widespread distribution of the Cys26-cystatin D encoding allele in the population. The inhibitory properties displayed by cystatin D suggest that it has a function in saliva as inhibitor of either endogenous or exogenous enzymes with cathepsin S- or H-like properties.
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PMID:Structural and functional characterization of two allelic variants of human cystatin D sharing a characteristic inhibition spectrum against mammalian cysteine proteinases. 808 19

Sertoli cells were shown to synthesize and secrete cystatin C, a potent inhibitor of cysteine proteases. The evidence for this observation was obtained from protein sequencing. Western analysis using antiserum specific to cystatin C, and immunoprecipitation of 35S protein secreted by cultured cells. The Western analysis with an antiserum to human cystatin C showed that cultured Sertoli cells secrete three previously reported immunoreactive forms of cystatin C: a predominant pair of proteins at 13-14 kDa and a less abundant 20-kDa protein. Immunohistochemical localization of cystatin C in sections of rat testes showed intense staining in Sertoli cells; no immunoreactivity was observed in spermatogonia or spermatocytes. A cDNA fragment for rat cystatin C was obtained by use of the polymerase chain reaction and was used as a probe in Northern analyses to examine the steady-state levels of cystatin C mRNA in intact testes and in Sertoli and spermatogenic cells. Sertoli cells contained a 700-nucleotide cystatin C transcript, and a mixed population of spermatids and spermatocytes contained a 550-nucleotide transcript. Analysis of RNA from purified spermatogenic cells revealed that round and condensing spermatids contained the 550-nucleotide transcript, while pachytene spermatocytes contained a smaller 500-nucleotide transcript. The 700-nucleotide transcript was present in testes isolated from rats of 5-79 days of age, the 500-nucleotide transcript was detected initially in testes from 24-day-old rats, and the 550-nucleotide transcript was detected initially at 35 days of age. Both the 500- and 550-nucleotide transcripts increased in abundance until 50 days of age. RNA from stage-synchronized testes showed that steady-state levels of both the 550- and 700-nucleotide transcripts were lowest in stages VI-VII of the cycle. These data suggest that the role of cystatin C in the testis may be to inhibit the proteolytic activity of the cysteine protease cathepsin L in all stages except stages VI-VII.
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PMID:Sertoli cell and germ cell cystatin C: stage-dependent expression of two distinct messenger ribonucleic acid transcripts in rat testes. 828 70


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